CN103361299B - The method of separate stem cells and test kit - Google Patents
The method of separate stem cells and test kit Download PDFInfo
- Publication number
- CN103361299B CN103361299B CN201210096966.0A CN201210096966A CN103361299B CN 103361299 B CN103361299 B CN 103361299B CN 201210096966 A CN201210096966 A CN 201210096966A CN 103361299 B CN103361299 B CN 103361299B
- Authority
- CN
- China
- Prior art keywords
- stem cell
- solution
- cell
- red corpuscle
- balanced salt
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The present invention relates to method and the test kit of separate stem cells.Wherein, the present invention proposes a kind of composition for separating of stem cell, it comprises: Schering AG); And balanced salt solution, wherein, described Schering AG) is dissolved in described balanced salt solution.Utilize said composition, can effective separate stem cells.
Description
Technical field
The present invention relates to field of biomedicine technology, especially method and the test kit of separate stem cells is related to, more specifically, the present invention relates to the purposes of Schering AG) in preparation stem cell refined solution, composition, the method for separate stem cells, the test kit of separate stem cells and the purposes of this test kit in separate stem cells for separating of stem cell.
Background technology
Containing a large amount of hematopoietic cells and all kinds of stem cell and a large amount of mature cells in the marrow of people and Cord blood.The white corpuscle of each lineage stem cells and maturation is karyocyte.Comprise multiple stem cell and list/syncyte in karyocyte, also have lymphocyte and mesenchymal cell etc.
The separation method of external erythrocyte and karyocyte comprises machine centrifuging and settling process.Machine centrifuging, with the separating effect of its totally closed operation and stability and safety, is widely used abroad.But it is because equipment price is expensive, domestic still difficult universal at present.Also has multiple method in addition: paramagnetic particle method, immunofluorescence analysis, Flow cytometry etc.The common problem of these methods is: the stem cell 1) obtained is impure, 2) using inconvenient, involve great expense, 3) technical equipment requires high, operation requires high to technician.
Thus, the technology of current separate stem cells still haves much room for improvement.
Summary of the invention
The present invention is intended at least to solve one of technical problem existed in prior art.For this reason, one object of the present invention be to propose a kind of can the means of effective separate stem cells.
The present invention completes based on the following discovery of contriver: the soluble in water and damping fluid of Schering AG), concentration can reach 80 % by weight, and the solution of Schering AG) can through autoclaving, and nontoxic low viscosity, does not affect Premeabilisation of cells pressure.Contriver is surprised to find, and when Schering AG) is applied to refined solution as density gradient centrifugation separating medium, can be widely used in the various types of cell of purifying, subcellular organelle and macromolecule complex, efficiency is obviously better than Percoll.
For this reason, in a first aspect of the present invention, the present invention proposes the purposes of Schering AG) in preparation stem cell refined solution.By Schering AG) being applied to preparation stem cell refined solution, the efficiency of separate stem cells effectively can be improved.
In a second aspect of the present invention, the present invention proposes a kind of composition for separating of stem cell.According to embodiments of the invention, said composition comprises: Schering AG); And balanced salt solution, wherein, described Schering AG) is dissolved in described balanced salt solution.Utilize said composition as stem cell refined solution, can effectively improve from the efficiency containing separate stem cells the biological sample of stem cell.
In a third aspect of the present invention, the present invention proposes a kind of method of separate stem cells.According to embodiments of the invention, the method comprises following: mixed with red corpuscle parting liquid by the biological specimen containing stem cell, and standing sedimentation is to obtain the upper strata enchylema containing stem cell; By the described upper strata enchylema centrifugal concentrating containing stem cell, obtain cell precipitation; With the first stem cell refined solution, described cell precipitation is carried out resuspension, obtain suspension; Described suspension is joined in the second stem cell refined solution centrifugal, and collect stem cell, wherein, described red corpuscle parting liquid is the methylated cellulose aqueous solution of 0.2-0.7 % by weight, and the composition of described first stem cell refined solution to be balanced salt solution and described second stem cell refined solution be foregoing separate stem cells (namely comprises Schering AG); And balanced salt solution, wherein, described Schering AG) is dissolved in described balanced salt solution).Contriver finds the method by adopting above-mentioned separate stem cells, effectively can improve the efficiency of separate stem cells.
In a fourth aspect of the present invention, the present invention proposes a kind of test kit for separating of stem cell.According to embodiments of the invention, this test kit comprises: red corpuscle parting liquid; First stem cell refined solution; And the second stem cell refined solution, wherein, described red corpuscle parting liquid is the methylated cellulose aqueous solution of 0.2-0.7 % by weight, the preferably methylated cellulose aqueous solution of 0.5 % by weight, the composition of described first stem cell refined solution to be balanced salt solution and described second stem cell refined solution be foregoing separate stem cells (namely comprises Schering AG); And balanced salt solution, wherein, described Schering AG) is dissolved in described balanced salt solution).Utilize this test kit, effectively can implement the method for aforementioned separate stem cells, thus, effectively can improve the efficiency of separate stem cells.
Additional aspect of the present invention and advantage will part provide in the following description, and part will become obvious from the following description, or be recognized by practice of the present invention.
Accompanying drawing explanation
Above-mentioned and/or additional aspect of the present invention and advantage will become obvious and easy understand from accompanying drawing below combining to the description of embodiment, wherein:
Fig. 1 is showing the schematic flow sheet of the method according to embodiments of the invention separate stem cells.
Embodiment
Be described below in detail embodiments of the invention, the example of described embodiment is shown in the drawings, and wherein same or similar label represents same or similar element or has element that is identical or similar functions from start to finish.Being exemplary below by the embodiment be described with reference to the drawings, only for explaining the present invention, and can not limitation of the present invention being interpreted as.
The present invention completes based on the following discovery of contriver: existing gradient centrifugation reagent such as Percoll, Filcoll solution be applied to purifying karyocyte especially stem cell time, efficiency is lower.Due to cell in vitro the time long, the impact of temperature and reagent is subject in sepn process, the recovery rate of nucleated cell that prior art obtains can only maintain 80-90%, and erythrocytic survival rate can reach about 4%, and due to prior art in vitro the operating time more than 2 hours, can greatly impact obtain the differentiation versatility of stem cell, reduce the differentiation potential of stem cell.Schering AG) (Iohexol, chemical name: Ominipaque Solution, trade(brand)name: Nycodenz, CAS no.66108-95-0) is a kind of molecular weight is 821, and density is 2.1g/ml, is non-ionic type, compound to cytotoxic.Contriver finds Schering AG) to be effectively applied to being separated or purification of the various biological substances such as cell, organoid, biomacromolecule and virus, and Schering AG) is stablized at high temperature under high pressure, thus can carry out autoclave sterilization to the solution be made into by Schering AG), thus Schering AG) is suitable for the gradient centrifugation reagent as separate stem cells.
In a first aspect of the present invention, the present invention proposes the purposes of Schering AG) in preparation stem cell refined solution.By utilizing Schering AG) to prepare the stem cell refined solution obtained, can, effectively for separating of purifying stem cell, especially be applicable to from spinal fluid and Cord blood separation and purification stem cell, preferably from anti-freezing spinal fluid and Cord blood.
In a second aspect of the present invention, the present invention proposes a kind of composition for separating of stem cell.According to embodiments of the invention, should be the solution of Schering AG) in balanced salt solution for separating of the composition of stem cell, namely the composition of this separate stem cells comprises Schering AG) and balanced salt solution, and wherein said Schering AG) is dissolved in described balanced salt solution.Can using said composition as gradient centrifugation reagent, effectively by gradient centrifugation from containing separate stem cells the sample of stem cell.
According to embodiments of the invention, the type of balanced salt solution is also not particularly limited, as long as it can maintain Premeabilisation of cells pressure balanced.According to one embodiment of present invention, the balanced salt solution that can adopt is at least one of HBSS (Hank ' s Balanced Salt Solution, Hank ' s balanced salt solution) and GBSS (GEY ' S balanced salt solution).Preferred balanced salt solution is HBSS.According to embodiments of the invention, the pH of said composition is 7-8.Thus, can separate stem cells effectively further.
According to embodiments of the invention, the concentration of Schering AG) is also not particularly limited.According to one embodiment of present invention, every 100ml balanced salt solution contains 8-15g Schering AG), preferred 13g Schering AG).Thus, the efficiency by gradient centrifugation separate stem cells can be improved further.According to embodiments of the invention, the composition containing Schering AG) in salt can be balanced easily, such as can by a certain amount of Schering AG) such as 13 grams of Schering AG)s and 100mlHBSS solution (can be obtained by commercial means, such as can purchased from invitrogen company), and stir certain hour, such as stir more than 1 hour, keep in Dark Place in 4 DEG C afterwards, for subsequent use.
In addition according to embodiments of the invention, in the composition, cell growth factor can also be comprised further, preferably comprise the human alkaline fibroblast somatomedin of 10-30ng/ml and the human stem cell growth-α of 10-30ng/ml further.Thus, contriver finds, by adding somatomedin in the composition, especially stem cell factor, can effectively improve stem cell in vitro survival rate, and can maintain the differentiation potential of stem cell.
In a third aspect of the present invention, the present invention proposes a kind of method of separate stem cells.With reference to figure 1, the method for this separate stem cells comprises the following steps:
First, separating red corpuscle.According to embodiments of the invention, the biological specimen containing stem cell can be passed through to mix with red corpuscle parting liquid, and standing sedimentation, make the lower floor of erythrocyte sedimentation liquid, and obtain the upper strata enchylema containing stem cell.The type of term " red corpuscle parting liquid " used here is also not particularly limited, as long as fully can be separated with the karyocyte comprising stem cell by red corpuscle by sedimentation.According to one embodiment of present invention, the red corpuscle parting liquid of employing is the methylated cellulose aqueous solution of 0.2-0.7 % by weight, preferably the methylated cellulose aqueous solution of 0.5 % by weight.According to embodiments of the invention, the settling time is also not particularly limited, and according to a particular embodiment of the invention, can implement sedimentation 45-60 minute.Thus, red corpuscle can be effectively made fully to be separated with the karyocyte comprising stem cell.Thus the upper strata enchylema obtained containing stem cell.According to embodiments of the invention, the type of the biological specimen containing stem cell is also not particularly limited, and can be any biological specimen containing stem cell be separated from any position of organism such as human body.According to one embodiment of present invention, the biological specimen containing stem cell can for being selected from least one of spinal fluid and Cord blood, preferred anti-freezing spinal fluid or Cord blood.Thus, a large amount of stem cells can be obtained, especially mescenchymal stem cell.According to embodiments of the invention, the blending ratio of the biological specimen containing stem cell and red corpuscle parting liquid is also not particularly limited.According to one embodiment of present invention, select such blending ratio, make final mixed solution contain the methylcellulose gum of 0.1 % by weight.Such as, for anti-freezing spinal fluid or Cord blood, according to the volume ratio of 4: 1, by anti-freezing spinal fluid or Cord blood with 0.5 % by weight methylated cellulose aqueous solution (red corpuscle parting liquid) mix.Thus, the efficiency of separating red corpuscle can be improved further, thus improve the efficiency of final separate stem cells further.According to embodiments of the invention, sedimentation can be repeated containing upper strata enchylema and remove erythrocytic operation, so that the red corpuscle that removing is remaining be further separated.
Next, collect upper strata enchylema, and the upper strata enchylema this being contained stem cell carries out centrifugal concentrating, thus obtain cell precipitation.And then utilize the first stem cell refined solution that obtained cell precipitation is carried out resuspension.It should be noted that, term " first " used in the present invention, " second " are only used to distinguish, and express or imply the difference of order between it and importance never in any form.In this article, the first stem cell refined solution adopted is at least one being selected from balanced salt solution and cell culture fluid.According to embodiments of the invention, the type of balanced salt solution and cell culture fluid is also not particularly limited, the example of the balanced salt solution that can adopt includes but not limited to GBSS solution (being sometimes also directly called GBSS), HBSS solution (being sometimes also directly called HBSS), the balanced salt solution that preferably can adopt is HBSS, and the nutrient solution that can adopt is MEM nutrient solution.According to embodiments of the invention, the upper strata enchylema containing stem cell is carried out the condition of centrifugal concentrating and is not particularly limited.According to one embodiment of present invention, can adopt and within centrifugal 5 minutes under 1200rpm, realize effectively concentrating cell.Can adopt a certain amount of balanced salt solution, such as 10ml balanced salt solution (the first stem cell refined solution) carries out resuspension to obtained cell precipitation, to obtain the suspension containing stem cell.
Next, after obtaining the suspension containing stem cell, obtained suspension can be joined in the second cell purification liquid and carry out gradient centrifugation, so that separate stem cells.According to embodiments of the invention, second stem cell refined solution is foregoing composition, the i.e. solution of Schering AG) in balanced salt solution (in other words, the composition of separate stem cells comprises Schering AG) and balanced salt solution, and wherein said Schering AG) is dissolved in described balanced salt solution).Thus, effectively can pass through gradient centrifugation, by karyocyte and other its cellular segregation, thus be convenient to separate stem cells especially mescenchymal stem cell (being usually located at the middle part of centrifugate).According to embodiments of the invention, centrifugal condition is also not particularly limited.According to a particular embodiment of the invention, can adopt centrifugal 22 minutes of 4 DEG C of Gradients.Thus, can separate stem cells effectively further.About the second stem cell refined solution, before for separate stem cells composition described by all technical characteristic and advantage can be applicable to this, for simplicity, repeat no more.The second stem cell refined solution is utilized to carry out the condition of gradient centrifugation and be not particularly limited.
According to embodiments of the invention, after obtaining stem cell, cell treatment solution can be adopted, obtained stem cell is cleaned.According to embodiments of the invention, the cell treatment solution that can adopt is at least one of physiological saline and phosphate buffered saline buffer.According to specific examples of the present invention, cell treatment solution can be used, at least twice is cleaned to obtained stem cell, thus, the purity of obtained stem cell can be ensured.According to embodiments of the invention, the type of cell treatment solution is also not particularly limited, can for being selected from least one of phosphoric acid buffer and physiological saline.Thus, the efficiency that stem cell is cleaned can be improved further.It should be noted that, phraseology used in this article " is selected from least one of phosphate buffered saline buffer and physiological saline " and refers to, namely can be used alone phosphate buffered saline buffer, also can be used alone physiological saline, the combination of phosphate buffered saline buffer and physiological saline can also be used.Here used " combination of phosphoric acid salt and physiological saline " should be interpreted broadly, can be used for cleaning stem cell after phosphate buffered saline buffer is mixed with physiological saline, also can be use phosphate buffered saline buffer and physiological saline to clean stem cell respectively, wherein, if use phosphate buffered saline buffer and physiological saline to clean stem cell respectively, then sequencing being not particularly limited, first can use phosphate buffered saline buffer, physiological saline is used to clean stem cell afterwards, also first physiological saline can be used, phosphate buffered saline buffer is used to clean stem cell afterwards.
According to embodiments of the invention, one of at least can comprising further of red corpuscle parting liquid adopted in the present invention, the first stem cell refined solution, the second stem cell refined solution and cell treatment solution: the human alkaline fibroblast somatomedin of 10-30ng/ml and the human stem cell growth-α of 10-30ng/ml.Preferred red corpuscle parting liquid, the first stem cell refined solution, the second stem cell refined solution and cell treatment solution comprise all further: the human alkaline fibroblast somatomedin of 10-30ng/ml and the human stem cell growth-α of 10-30ng/ml.Thus, contriver finds, by adding somatomedin, especially stem cell factor, can effectively improve stem cell in vitro survival rate, and can maintain the differentiation potential of stem cell.
Utilize the method according to the separate stem cells of the embodiment of the present invention, can realize being separated the stem cell in spinal fluid and Cord blood, the stem cell survival be separated reaches more than 99%, and stem cell yield reaches 90%.According to embodiments of the invention, aforesaid operations all can operate in Biohazard Safety Equipment according to working specification, and separation can not be polluted because of reagent.Simultaneously according to according to the isolated stem cell surface of the method for the embodiment of the present invention without any marker, do not change the biological activity of cell.In addition, according to the method for the embodiment of the present invention, owing to adding somatomedin in the reagent in sepn process, thus, stem cell improves survival rate and maintains the versatility of stem cell in sepn process.
In a fourth aspect of the present invention, the present invention proposes a kind of test kit for separating of stem cell.According to embodiments of the invention, this test kit comprises: red corpuscle parting liquid, the first stem cell refined solution and the second stem cell refined solution.According to embodiments of the invention, in this test kit, can further include cell treatment solution.
According to embodiments of the invention, red corpuscle parting liquid is the methylated cellulose aqueous solution of 0.2-0.7 % by weight, preferably the methylated cellulose aqueous solution of 0.5 % by weight, can effectively for by settlement separate removing red corpuscle.According to embodiments of the invention, the first stem cell refined solution is at least one being selected from balanced salt solution and cell culture fluid.According to embodiments of the invention, the type of balanced salt solution and cell culture fluid is also not particularly limited, according to one embodiment of present invention, the balanced salt solution that can adopt is at least one of HBSS (Hank ' sBalanced Salt Solution, Hank ' s balanced salt solution) and GBSS (GEY ' S balanced salt solution).Preferred balanced salt solution is HBSS, and the nutrient solution that can adopt is MEM nutrient solution.Effectively can carry out resuspension, to carry out down-stream to the cell precipitation containing stem cell of centrifugal concentrating.According to embodiments of the invention, second stem cell refined solution is foregoing composition, the i.e. solution of Schering AG) in balanced salt solution (in other words, the composition of separate stem cells comprises Schering AG) and balanced salt solution, and wherein said Schering AG) is dissolved in described balanced salt solution).Thus, effectively can pass through gradient centrifugation, by karyocyte and other cellular segregation, thus be convenient to separate stem cells especially mescenchymal stem cell (being usually located at the middle part of centrifugate).About the second stem cell refined solution, before for separate stem cells composition described by all technical characteristic and advantage can be applicable to this, for simplicity, repeat no more.According to embodiments of the invention, cell treatment solution is at least one of phosphoric acid buffer and physiological saline.Thus, this cell treatment solution can be utilized easily to clean being separated the stem cell obtained.Thus, utilize this test kit, effectively can implement the method for aforementioned separate stem cells, thus can effectively from the biological specimen separate stem cells containing stem cell.
According to embodiments of the invention, one of at least can comprising further of red corpuscle parting liquid adopted in the present invention, the first stem cell refined solution, the second stem cell refined solution and cell treatment solution: the human alkaline fibroblast somatomedin of 10-30ng/ml and the human stem cell growth-α of 10-30ng/ml.Preferred red corpuscle parting liquid, the first stem cell refined solution, the second stem cell refined solution and cell treatment solution comprise all further: the human alkaline fibroblast somatomedin of 10-30ng/ml and the human stem cell growth-α of 10-30ng/ml.Thus, contriver finds, by adding somatomedin, especially stem cell factor, can effectively improve stem cell in vitro survival rate, and can maintain the differentiation potential of stem cell.According to embodiments of the invention, described red corpuscle parting liquid, the first stem cell refined solution and the second stem cell refined solution are separately positioned in different containers.Thus, can this test kit separate stem cells easy to use.Test kit of the present invention can suitability for industrialized production easily, utilizes this test kit, can obtain high quality and highly purified mescenchymal stem cell easily for scientific research or clinical treatment.
Thus, in a fifth aspect of the present invention, the present invention proposes the purposes of mentioned reagent box in separate stem cells.Adopt the test kit of the embodiment of the present invention, effectively can implement the method for the separate stem cells of the embodiment of the present invention.Whole feature and advantage about this separation method are all applicable to herein, for the sake of simplicity, repeat no more.
Below with reference to specific embodiment, the present invention will be described, it should be noted that, these embodiments are only illustrative, and can not be interpreted as limitation of the present invention.
If do not specialize, the conventional means that the technique means adopted in embodiment is well known to those skilled in the art, can carry out with reference to " Molecular Cloning: A Laboratory guide " third edition or related products, the reagent adopted and product be also can business obtain.The various process do not described in detail and method are ordinary methods as known in the art, source, the trade(brand)name of agents useful for same and be necessary to list its moiety person, all indicate when occurring first, identical reagent used if no special instructions thereafter, all identical with the content indicated first.
Embodiment 1 (general material and general method)
Test kit:
Reagent A: red corpuscle parting liquid
0.5-0.6% methylcellulose gum (MC) (FLUN K company of Switzerland).
Methylcellulose gum average viscosity is 4,000cP, first, with 4 DEG C of normal saline 0.4-0.6 % by weight methocel solutions of precooling, puts 4 DEG C and spends the night, then vibration shakes up repeatedly, dissolves completely to methylcellulose gum.By obtained methocel solution autoclave sterilization 15 minutes under 0.1MPa, after being cooled to 4 DEG C, use forced oscillation again.In water white transparency shape after methylcellulose gum dissolves completely, pH 7-8, puts 4 DEG C and saves backup.
Reagent B: stem cell refined solution I
HBSS solution (purchased from Invitrogen company) or GBSS solution or MEM nutrient solution
Reagent C: stem cell refined solution II
8-15 % by weight Schering AG) solution: by the Schering AG) (Nycodenz) of predetermined amount (8-15g), mixes with 100mlHBSS solution (Invitrogen company), and stirs more than 1 hour, finally keep in Dark Place in 4 DEG C.pH 7-8。
Schering AG) (Nycodenz) is the pulvis of a kind of white, high-hydrophilic, and chemical composition is Schering AG) (Iohexol, CAS no.66108-95-0), can be made into the nonionic density gradient separation liquid of Almightiness type.
The molecular weight of Schering AG) (Nycodenz) is 821, and density is 2.1g/ml, is non-ionic type, compound to cytotoxic, can be used for separation or the purification of the various biological substances such as cell, organoid, biomacromolecule, virus.With the parting liquid that Schering AG) (Nycodenz) is made into, the sterilizing of High Temperature High Pressure can be carried out.
Reagent D: cell treatment solution
Phosphoric acid buffer or physiological saline
After mentioned reagent is prepared, through 121 DEG C, after 1 hour high-temperature sterilization, test endotoxin content≤0.5EU/ml.Leave in respectively in test kit.Add somatomedin before use, in often kind of reagent, add human alkaline fibroblast somatomedin (hFGF2) that final concentration is 10-30ng/ml and the 10-30ng/ml human stem cell growth-α (former stem cell factor) that final concentration is.
General method
I, separating red corpuscle:
Method 1: the anti-freezing bone marrow fluid gathered or Cord blood and red corpuscle parting liquid are mixed, is placed in 100ml Open system device, under room temperature in Bechtop, hang and leave standstill, sedimentation 45-60 minute, releases the red corpuscle of lower floor completely, collects upper strata enchylema centrifugal concentrating.With the cell precipitation that stem cell refined solution I resuspension centrifugal concentrating obtains.
Method 2: the anti-freezing bone marrow fluid gathered or Cord blood and red corpuscle parting liquid are mixed, can, by sample in predetermined ratio, directly add in red corpuscle parting liquid container, under room temperature in Bechtop, leave standstill, sedimentation 45-60 minute, collect upper strata enchylema centrifugal concentrating.With the cell precipitation that stem cell refined solution I resuspension centrifugal concentrating obtains.
II, gradient centrifugation separate stem cells:
By the suspension that the cell precipitation utilizing cell purification liquid I resuspension centrifugal concentrating to obtain obtains, slowly join on stem cell refined solution II, drip gently, to avoid confusing boundary layer.Then by centrifugal for centrifuge tube 22 minutes, collect middle level oyster white layer, repeatedly clean cell 2 times with cell treatment solution, obtain stem cell.
Embodiment 2
Reagent A: red corpuscle parting liquid
20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) is added in 0.5% methylcellulose gum.
Reagent B: stem cell refined solution I
20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) is added in HBSS liquid (purchased from Invitrogen company).
Reagent C: stem cell refined solution II
13%Nycodenz solution: Nycodenz 13g, HBSS solution (invitrogen company) 100ml, stir > 1 hour, add 20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor).
Reagent D: cell treatment solution
20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) is added in phosphoric acid buffer.
The 80ml anti-freezing bone marrow fluid gathered or Cord blood and 20ml 0.5 % by weight red corpuscle parting liquid are pressed 4: 1 (v: v) mix, red corpuscle parting liquid final concentration is made to be 0.1 % by weight (methylcellulose gum, lower same), and be placed in 100ml Open system device.Under room temperature, in Bechtop, hang and leave standstill, sedimentation 45-60 minute, release the red corpuscle of lower floor completely, make red corpuscle, white corpuscle demarcate liquid level be in transfusion device under the diameter that connects be the translucent silica gel tubing upper end of 5mm, then sedimentation 5 minutes, slowly release lower floor's red corpuscle, collect upper strata enchylema 1200rpm5 minute centrifugal concentrating.With 10ml reagent B diluting cells, the cell suspending liquid of collection is slowly joined in reagent C.900g, centrifugal 22 minutes of 4 DEG C of Gradients, collect middle level oyster white layer, repeatedly clean cell 2 times with cell treatment solution.Finally use 5ml cell treatment solution diluting cells for subsequent use.Get 5 microlitre stem cell samples, detect cell survival rate by tetrabromophenol sulfonphthalein, reach 99%.Get 5 microlitre stem cell samples again, carry out cell counting, conclusion is that every 80ml marrow or Cord blood separation obtain at least 4 × 10
7individual stem cell
Embodiment 3
Reagent A: red corpuscle parting liquid
20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) is added in 0.5% methylcellulose gum.
Reagent B: stem cell refined solution I
20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) is added in HBSS liquid (purchased from Invitrogen company).
Reagent C: stem cell refined solution II
13%Nycodenz solution: Nycodenz 13g, HBSS solution (invitrogen company) 100ml, stir > 1 hour, add 20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor).
Reagent D: cell treatment solution
20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) is added in physiological saline.
By in the 80ml anti-freezing bone marrow fluid of collection or Cord blood and 20ml reagent A bottle, under room temperature in Bechtop, leave standstill, sedimentation 45-60 minute, precipitates the red corpuscle of lower floor completely, collects upper strata enchylema, 1200rpm5 minute centrifugal concentrating.Dilute the cell mass of precipitation with 10ml reagent B, the cell suspending liquid of collection is slowly joined in reagent C.900g, centrifugal 22 minutes of 4 DEG C of Gradients, collect middle level oyster white layer, repeatedly clean cell 2 times by reagent D.For subsequent use with 5ml reagent D diluting cells.Get 5 microlitre tetrabromophenol sulfonphthaleins to detect cell survival rates and reach 99% and get 5 microlitres again and carry out cell counting.Conclusion is that every 80ml marrow or Cord blood separation obtain 4 × 10
7individual stem cell.
Embodiment 4
Reagent A: red corpuscle parting liquid
20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) is added in 0.5% methylcellulose gum.
Reagent B: stem cell refined solution I
20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) is added in GBSS liquid (purchased from Invitrogen company).
Reagent C: stem cell refined solution II
13%Nycodenz solution: Nycodenz 13g, GBSS solution (invitrogen company) 100ml, stir > 1 hour, add 20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor).
Reagent D: cell treatment solution
20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) is added in phosphoric acid buffer.
The 80ml anti-freezing bone marrow fluid gathered or Cord blood and 20ml 0.5 % by weight red corpuscle parting liquid are pressed 4: 1, and (v: v) mix makes red corpuscle parting liquid final concentration be 0.1 % by weight, and is placed in 100ml Open system device.Under room temperature, in Bechtop, hang and leave standstill, sedimentation 45-60 minute, release the red corpuscle of lower floor completely, make red corpuscle, white corpuscle demarcate liquid level be in transfusion device under the diameter that connects be the translucent silica gel tubing upper end of 5mm, then sedimentation 5 minutes, slowly release lower floor's red corpuscle, collect upper strata enchylema 1200rpm 5 minutes centrifugal concentratings.With 10ml reagent B diluting cells, the cell suspending liquid of collection is slowly joined in reagent C.900g, centrifugal 22 minutes of 4 DEG C of Gradients, collect middle level oyster white layer, repeatedly clean cell 2 times with cell treatment solution.Finally use 5ml cell treatment solution diluting cells for subsequent use.Get 5 microlitre stem cell samples, reach 98%. and get 5 microlitre stem cell samples again, carry out cell counting with tetrabromophenol sulfonphthalein detection cell survival rate, conclusion is that every 80ml marrow or Cord blood separation obtain 4 × 10
7individual stem cell.
Embodiment 5
Reagent A: red corpuscle parting liquid
20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) is added in 0.5% methylcellulose gum.
Reagent B: stem cell refined solution I
20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) is added in GBSS liquid (purchased from Invitrogen company).
Reagent C: stem cell refined solution II
13%Nycodenz solution: Nycodenz 13g, GBSS solution (invitrogen company) 100ml, stir > 1 hour, add 20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor).
Reagent D: cell treatment solution
20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) is added in physiological saline.
The 80ml anti-freezing bone marrow fluid gathered or Cord blood and 20ml 0.5 % by weight red corpuscle parting liquid are pressed 4: 1, and (v: v) mix makes red corpuscle parting liquid final concentration be 0.1 % by weight, and is placed in 100ml Open system device.Under room temperature, in Bechtop, hang and leave standstill, sedimentation 45-60 minute, release the red corpuscle of lower floor completely, make red corpuscle, white corpuscle demarcate liquid level be in transfusion device under the diameter that connects be the translucent silica gel tubing upper end of 5mm, then sedimentation 5 minutes, slowly release lower floor's red corpuscle, collect upper strata enchylema 1200rpm5 minute centrifugal concentrating.With 10ml reagent B diluting cells, the cell suspending liquid of collection is slowly joined in reagent C.900g, centrifugal 22 minutes of 4 DEG C of Gradients, collect middle level oyster white layer, repeatedly clean cell 2 times with cell treatment solution.Finally use 5ml cell treatment solution diluting cells for subsequent use.Get 5 microlitre stem cell samples, reach 98%. and get 5 microlitre stem cell samples again, carry out cell counting with tetrabromophenol sulfonphthalein detection cell survival rate, conclusion is that every 80ml marrow or Cord blood separation obtain 4 × 10
7individual stem cell.
Embodiment 6
Reagent A: red corpuscle parting liquid
20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) is added in 0.5% methylcellulose gum.
Reagent B: stem cell refined solution I
20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) is added in MEM nutrient solution (purchased from Invitrogen company).
Reagent C: stem cell refined solution II
13%Nycodenz solution: Nycodenz 13g, HBSS solution (invitrogen company) 100ml, stir > 1 hour, add 20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor).
Reagent D: cell treatment solution
20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) is added in phosphoric acid buffer.
The 80ml anti-freezing bone marrow fluid gathered or Cord blood and 20ml0.5 % by weight red corpuscle parting liquid are pressed 4: 1, and (v: v) mix makes red corpuscle parting liquid final concentration be 0.1 % by weight, and is placed in 100ml Open system device.Under room temperature, in Bechtop, hang and leave standstill, sedimentation 45-60 minute, release the red corpuscle of lower floor completely, make red corpuscle, white corpuscle demarcate liquid level be in transfusion device under the diameter that connects be the translucent silica gel tubing upper end of 5mm, then sedimentation 5 minutes, slowly release lower floor's red corpuscle, collect upper strata enchylema 1200rpm5 minute centrifugal concentrating.With 10ml reagent B diluting cells, the cell suspending liquid of collection is slowly joined in reagent C.900g, centrifugal 22 minutes of 4 DEG C of Gradients, collect middle level oyster white layer, repeatedly clean cell 2 times with cell treatment solution.Finally use 5ml cell treatment solution diluting cells for subsequent use.Get 5 microlitre stem cell samples, reach 99%. and get 5 microlitre stem cell samples again, carry out cell counting with tetrabromophenol sulfonphthalein detection cell survival rate, conclusion is that every 80ml marrow or Cord blood separation obtain 4 × 10
7individual stem cell.
Embodiment 7
Reagent A: red corpuscle parting liquid
20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) is added in 0.5% methylcellulose gum.
Reagent B: stem cell refined solution I
20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) is added in MEM nutrient solution (purchased from Invitrogen company).
Reagent C: stem cell refined solution II
13%Nycodenz solution: Nycodenz 13g, HBSS solution (invitrogen company) 100ml, stir > 1 hour, add 20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor).
Reagent D: cell treatment solution
20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) is added in physiological saline.
The 80ml anti-freezing bone marrow fluid gathered or Cord blood and 20ml0.5 % by weight red corpuscle parting liquid are pressed 4: 1, and (v: v) mix makes red corpuscle parting liquid final concentration be 0.1 % by weight, and is placed in 100ml Open system device.Under room temperature, in Bechtop, hang and leave standstill, sedimentation 45-60 minute, release the red corpuscle of lower floor completely, make red corpuscle, white corpuscle demarcate liquid level be in transfusion device under the diameter that connects be the translucent silica gel tubing upper end of 5mm, then sedimentation 5 minutes, slowly release lower floor's red corpuscle, collect upper strata enchylema 1200rpm5 minute centrifugal concentrating.With 10ml reagent B diluting cells, the cell suspending liquid of collection is slowly joined in reagent C.900g, centrifugal 22 minutes of 4 DEG C of Gradients, collect middle level oyster white layer, repeatedly clean cell 2 times with cell treatment solution.Finally use 5ml cell treatment solution diluting cells for subsequent use.Get 5 microlitre stem cell samples, reach 99%. and get 5 microlitre stem cell samples again, carry out cell counting with tetrabromophenol sulfonphthalein detection cell survival rate, conclusion is that every 80ml marrow or Cord blood separation obtain 4 × 10
7individual stem cell.
Embodiment 8
Reagent A: red corpuscle parting liquid
20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) is added in 0.5% methylcellulose gum.
Reagent B: stem cell refined solution I
20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) is added in MEM nutrient solution (purchased from Invitrogen company).
Reagent C: stem cell refined solution II
8%Nycodenz solution: Nycodenz 8g, HBSS solution (invitrogen company) 100ml, stir > 1 hour, add 20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor).
Reagent D: cell treatment solution
20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) is added in physiological saline.
The 80ml anti-freezing bone marrow fluid gathered or Cord blood and 20ml0.5 % by weight red corpuscle parting liquid are pressed 4: 1, and (v: v) mix makes red corpuscle parting liquid final concentration be 0.1 % by weight, and is placed in 100ml Open system device.Under room temperature, in Bechtop, hang and leave standstill, sedimentation 45-60 minute, release the red corpuscle of lower floor completely, make red corpuscle, white corpuscle demarcate liquid level be in transfusion device under the diameter that connects be the translucent silica gel tubing upper end of 5mm, then sedimentation 5 minutes, slowly release lower floor's red corpuscle, collect upper strata enchylema 1200rpm 5 minutes centrifugal concentratings.With 10ml reagent B diluting cells, the cell suspending liquid of collection is slowly joined in reagent C.900g, centrifugal 22 minutes of 4 DEG C of Gradients, collect middle level oyster white layer, repeatedly clean cell 2 times with cell treatment solution.Finally use 5ml cell treatment solution diluting cells for subsequent use.Get 5 microlitre stem cell samples, reach 99%. and get 5 microlitre stem cell samples again, carry out cell counting with tetrabromophenol sulfonphthalein detection cell survival rate, conclusion is that every 80ml marrow or Cord blood separation obtain 4 × 10
7individual stem cell.
Embodiment 9
Reagent A: red corpuscle parting liquid
20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) is added in 0.5% methylcellulose gum.
Reagent B: stem cell refined solution I
20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) is added in MEM nutrient solution (purchased from Invitrogen company).
Reagent C: stem cell refined solution II
11%Nycodenz solution: Nycodenz 11g, HBSS solution (invitrogen company) 100ml, stir > 1 hour, add 20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor).
Reagent D: cell treatment solution
20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) is added in physiological saline.
The 80ml anti-freezing bone marrow fluid gathered or Cord blood and 20ml0.5 % by weight red corpuscle parting liquid are pressed 4: 1, and (v: v) mix makes red corpuscle parting liquid final concentration be 0.1 % by weight, and is placed in 100ml Open system device.Under room temperature, in Bechtop, hang and leave standstill, sedimentation 45-60 minute, release the red corpuscle of lower floor completely, make red corpuscle, white corpuscle demarcate liquid level be in transfusion device under the diameter that connects be the translucent silica gel tubing upper end of 5mm, then sedimentation 5 minutes, slowly release lower floor's red corpuscle, collect upper strata enchylema 1200rpm5 minute centrifugal concentrating.With 10ml reagent B diluting cells, the cell suspending liquid of collection is slowly joined in reagent C.900g, centrifugal 22 minutes of 4 DEG C of Gradients, collect middle level oyster white layer, repeatedly clean cell 2 times with cell treatment solution.Finally use 5ml cell treatment solution diluting cells for subsequent use.Get 5 microlitre stem cell samples, reach 99%. and get 5 microlitre stem cell samples again, carry out cell counting with tetrabromophenol sulfonphthalein detection cell survival rate, conclusion is that every 80ml marrow or Cord blood separation obtain 4 × 10
7individual stem cell.
Embodiment 10
Reagent A: red corpuscle parting liquid
20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) is added in 0.5% methylcellulose gum.
Reagent B: stem cell refined solution I
20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) is added in MEM nutrient solution (purchased from Invitrogen company).
Reagent C: stem cell refined solution II
15%Nycodenz solution: Nycodenz 15g, HBSS solution (invitrogen company) 100ml, stir > 1 hour, add 20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor).
Reagent D: cell treatment solution
20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) is added in physiological saline.
The 80ml anti-freezing bone marrow fluid gathered or Cord blood and 20ml0.5 % by weight red corpuscle parting liquid are pressed 4: 1, and (v: v) mix makes red corpuscle parting liquid final concentration be 0.1 % by weight, and is placed in 100ml Open system device.Under room temperature, in Bechtop, hang and leave standstill, sedimentation 45-60 minute, release the red corpuscle of lower floor completely, make red corpuscle, white corpuscle demarcate liquid level be in transfusion device under the diameter that connects be the translucent silica gel tubing upper end of 5mm, then sedimentation 5 minutes, slowly release lower floor's red corpuscle, collect upper strata enchylema 1200rpm 5 minutes centrifugal concentratings.With 10ml reagent B diluting cells, the cell suspending liquid of collection is slowly joined in reagent C.900g, centrifugal 22 minutes of 4 DEG C of Gradients, collect middle level oyster white layer, repeatedly clean cell 2 times with cell treatment solution.Finally use 5ml cell treatment solution diluting cells for subsequent use.Get 5 microlitre stem cell samples, reach 99%. and get 5 microlitre stem cell samples again, carry out cell counting with tetrabromophenol sulfonphthalein detection cell survival rate, conclusion is that every 80ml marrow or Cord blood separation obtain 4 × 10
7individual stem cell.
Embodiment 11
Reagent A: red corpuscle parting liquid
20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) is added in 0.5% methylcellulose gum.
Reagent B: stem cell refined solution I
Add 20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) in HBSS (purchased from Invitrogen company), add phenol red.
Reagent C: stem cell refined solution II
13%Nycodenz solution: Nycodenz 13g, HBSS solution (invitrogen company) 100ml, stir > 1 hour, add 20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor), add phenol red.
Reagent D: cell treatment solution
20ng/ml human alkaline fibroblast somatomedin (hFGF2) and 20ng/ml human stem cell growth-α (former stem cell factor) is added in physiological saline.
The 80ml anti-freezing bone marrow fluid gathered or Cord blood and 20ml 0.5 % by weight red corpuscle parting liquid are pressed 4: 1, and (v: v) mix makes red corpuscle parting liquid final concentration be 0.1 % by weight, and is placed in 100ml Open system device.Under room temperature, in Bechtop, hang and leave standstill, sedimentation 45-60 minute, release the red corpuscle of lower floor completely, make red corpuscle, white corpuscle demarcate liquid level be in transfusion device under the diameter that connects be the translucent silica gel tubing upper end of 5mm, then sedimentation 5 minutes, slowly release lower floor's red corpuscle, collect upper strata enchylema 1200rpm5 minute centrifugal concentrating.With 10ml reagent B diluting cells, the cell suspending liquid of collection is slowly joined in reagent C.900g, centrifugal 22 minutes of 4 DEG C of Gradients, collect middle level oyster white layer, repeatedly clean cell 2 times with cell treatment solution.Finally use 5ml cell treatment solution diluting cells for subsequent use.Get 5 microlitre stem cell samples, reach 99%. and get 5 microlitre stem cell samples again, carry out cell counting with tetrabromophenol sulfonphthalein detection cell survival rate, conclusion is that every 80ml marrow or Cord blood separation obtain 4 × 10
7individual stem cell.
In the description of this specification sheets, specific features, structure, material or feature that the description of reference term " embodiment ", " some embodiments ", " example ", " concrete example " or " some examples " etc. means to describe in conjunction with this embodiment or example are contained at least one embodiment of the present invention or example.In this manual, identical embodiment or example are not necessarily referred to the schematic representation of above-mentioned term.And the specific features of description, structure, material or feature can combine in an appropriate manner in any one or more embodiment or example.
Although illustrate and describe embodiments of the invention, those having ordinary skill in the art will appreciate that: can carry out multiple change, amendment, replacement and modification to these embodiments when not departing from principle of the present invention and aim, scope of the present invention is by claim and equivalents thereof.
Claims (10)
1. a method for separate stem cells, is characterized in that, comprises the following steps:
Biological specimen containing stem cell is mixed with red corpuscle parting liquid, and standing sedimentation is to obtain the upper strata enchylema containing stem cell;
By the described upper strata enchylema centrifugal concentrating containing stem cell, obtain cell precipitation;
With the first stem cell refined solution, described cell precipitation is carried out resuspension, obtain suspension;
Described suspension to be joined in the second stem cell refined solution and centrifugal, to collect stem cell,
Wherein,
Described red corpuscle parting liquid is the methylated cellulose aqueous solution of 0.2-0.7 % by weight,
Described first stem cell refined solution is at least one being selected from balanced salt solution and cell culture fluid,
Described second stem cell refined solution comprises:
Schering AG); Balanced salt solution; The human alkaline fibroblast somatomedin of 20ng/ml and the human stem cell growth-α of 20ng/ml,
Wherein,
Described Schering AG) is dissolved in described balanced salt solution, and every 100ml balanced salt solution contains 8-15g Schering AG),
The pH of described second stem cell refined solution is 7-8.
2. method according to claim 1, is characterized in that, described every 100ml balanced salt solution contains 13g Schering AG).
3. method according to claim 1, is characterized in that, described red corpuscle parting liquid is the methylated cellulose aqueous solution of 0.5 % by weight.
4. method according to claim 1, is characterized in that, described balanced salt solution is at least one being selected from HBSS and GBSS, and described cell culture fluid is MEM nutrient solution.
5. method according to claim 4, is characterized in that, described balanced salt solution is HBSS.
6. method according to claim 1, is characterized in that, the described biological specimen containing stem cell is at least one being selected from spinal fluid and Cord blood.
7. method according to claim 6, is characterized in that, the described biological specimen containing stem cell is anti-freezing spinal fluid or Cord blood.
8. method according to claim 6, is characterized in that, the described biological specimen containing stem cell mixes according to volume ratio 4:1 with described red corpuscle centrifugate.
9. method according to claim 6, is characterized in that, comprises further, uses cell treatment solution to clean at least one times to obtained stem cell,
Wherein,
Described cell treatment solution is at least one being selected from phosphoric acid buffer and physiological saline.
10. method according to claim 9, is characterized in that, uses cell treatment solution to carry out twice cleaning to obtained stem cell.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210096966.0A CN103361299B (en) | 2012-04-01 | 2012-04-01 | The method of separate stem cells and test kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210096966.0A CN103361299B (en) | 2012-04-01 | 2012-04-01 | The method of separate stem cells and test kit |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103361299A CN103361299A (en) | 2013-10-23 |
| CN103361299B true CN103361299B (en) | 2015-09-30 |
Family
ID=49363566
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210096966.0A Active CN103361299B (en) | 2012-04-01 | 2012-04-01 | The method of separate stem cells and test kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103361299B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP6992385B2 (en) * | 2017-10-02 | 2022-01-13 | 株式会社島津製作所 | Separation medium for electrophoresis, reagent kit for electrophoresis, and electrophoresis method |
| CN110283782A (en) * | 2019-06-27 | 2019-09-27 | 昆明医科大学第二附属医院 | A kind of method for building up of human marrow mesenchymal stem cell strain and induction differentiation application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1728946A (en) * | 2002-07-19 | 2006-02-01 | 维斯塔治疗公司 | Method of obtaining viable human liver cells, including hepatic stem/progenitor cells |
| WO2007137769A2 (en) * | 2006-05-29 | 2007-12-06 | Eberhard-Karls-Universität Tübingen | Method for the in vitro preparation of living cells and/or cell accumulations from tissue |
-
2012
- 2012-04-01 CN CN201210096966.0A patent/CN103361299B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1728946A (en) * | 2002-07-19 | 2006-02-01 | 维斯塔治疗公司 | Method of obtaining viable human liver cells, including hepatic stem/progenitor cells |
| WO2007137769A2 (en) * | 2006-05-29 | 2007-12-06 | Eberhard-Karls-Universität Tübingen | Method for the in vitro preparation of living cells and/or cell accumulations from tissue |
Non-Patent Citations (2)
| Title |
|---|
| Iodixanol梯度离心法分离脂蛋白;党美林 等;《第七届海峡两岸心血管研讨会论文集》;20091231;54 * |
| 人脐带血间充质干细胞的体外分离、培养及鉴定;罗建蓉;《中国优秀硕士学位论文全文数据库2007年 医药卫生科技辑》;20070315(第3期);E059-10 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103361299A (en) | 2013-10-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US10119113B2 (en) | Systems for isolating and using clinically safe adipose derived regenerative cells | |
| AU2011250989B2 (en) | Cell-culture-bag | |
| JP4731556B2 (en) | System and method for isolating and using clinically safe adipose tissue-derived regenerative cells | |
| US9101926B2 (en) | Method for separating a sample into density specific fractions | |
| US9095798B2 (en) | Centrifuge separation method and apparatus using a medium density fluid | |
| US20080050814A1 (en) | Procurement, isolation and cryopreservation of fetal placental cells | |
| WO2007146105A2 (en) | Procurement, isolation and cryopreservation of fetal placental cells | |
| CN106244532A (en) | The preparation method of people source umbilical cord mesenchymal stem cells | |
| CA2745214A1 (en) | System and method for separating cells from body fluids | |
| JP2016507760A (en) | Liquid-liquid biological particle concentrator with a disposable fluid path | |
| CN104402993A (en) | Method for preparing human immunoglobulin for intravenous injection | |
| CN102321583B (en) | Method for separating umbilical cord blood hematopoietic stem cell by gradient centrifugation | |
| CN103361299B (en) | The method of separate stem cells and test kit | |
| CN102844121A (en) | Centrifuge tube | |
| US20080213819A1 (en) | Device for Preparing a Body Fluid for a Bacteriological Analysis | |
| CN102965339A (en) | Kit for treating human bone marrow, umbilical cord blood, and peripheral blood cells, and cell treatment method | |
| JP6452271B2 (en) | Serum preparation method | |
| CN102747034A (en) | Kit for in-vitro separation purification of karyocytes and use method thereof | |
| CN109652372A (en) | A kind of quick separating of human placenta source candidate stem cell, preparation method | |
| ES2525273T3 (en) | High security process for the preparation of purified stem cell fractions | |
| Schlinker | Automated, spinning membrane filtration for preparation of mobilized leukapheresis products for CD34+ cell selection | |
| KR20170138448A (en) | In vitro preservation of therapeutic cells | |
| WO2011087103A1 (en) | Proliferation promoter for mesenchymal stem cells, method for promoting proliferation of mesenchymal stem cells using same, and method for producing same | |
| Kumaratilake et al. | Purification of human monocytes/macrophages by adherence to cytodex microcarriers | |
| EP3143133A1 (en) | Isolation of adipose derived cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C53 | Correction of patent for invention or patent application | ||
| CB03 | Change of inventor or designer information |
Inventor after: Zhang Wenwei Inventor before: Zhang Wenwei Inventor before: Yang Guanghua |
|
| COR | Change of bibliographic data |
Free format text: CORRECT: INVENTOR; FROM: ZHANG WENWEI YANG GUANGHUA TO: ZHANG WENWEI |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20171206 Address after: No. 88 Jiangsu province 226601 City Branch Tongzhou Bay Jianghai Road No. 5 Building Patentee after: Jiangsu Yuan Sheng cell engineering science and Technology Development Co., Ltd. Address before: 201203 Shanghai City, Pudong New Area Zhangjiang road 377 Lane 144 Chenhui Patentee before: Zhang Wenwei |